Abstract

Choline is a water-soluble nutrient essential for human life. Gut microbial metabolism of choline results in the production of trimethylamine (TMA), which upon absorption by the host is converted in the liver to trimethylamine-N-oxide (TMAO). Recent studies revealed that TMAO exacerbates atherosclerosis in mice and positively correlates with the severity of this disease in humans. However, which microbes contribute to TMA production in the human gut, the extent to which host factors (e.g., genotype) and diet affect TMA production and colonization of these microbes, and the effects TMA-producing microbes have on the bioavailability of dietary choline remain largely unknown. We screened a collection of 79 sequenced human intestinal isolates encompassing the major phyla found in the human gut and identified nine strains capable of producing TMA from choline in vitro. Gnotobiotic mouse studies showed that TMAO accumulates in the serum of animals colonized with TMA-producing species, but not in the serum of animals colonized with intestinal isolates that do not generate TMA from choline in vitro. Remarkably, low levels of colonization by TMA-producing bacteria significantly reduced choline levels available to the host. This effect was more pronounced as the abundance of TMA-producing bacteria increased. Our findings provide a framework for designing strategies aimed at changing the representation or activity of TMA-producing bacteria in the human gut and suggest that the TMA-producing status of the gut microbiota should be considered when making recommendations about choline intake requirements for humans.

IMPORTANCE:

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and increased trimethylamine N-oxide (TMAO) levels have been causally linked with CVD development. This work identifies members of the human gut microbiota responsible for both the accumulation of trimethylamine (TMA), the precursor of the proatherogenic compound TMAO, and subsequent decreased choline bioavailability to the host. Understanding how to manipulate the representation and function of choline-consuming, TMA-producing species in the intestinal microbiota could potentially lead to novel means for preventing or treating atherosclerosis and choline deficiency-associated diseases.

Colonization with TMA-producing bacteria affects the levels of choline and TMAO in serum. (A) COPRO-Seq (community profiling by sequencing) analysis of cecal contents from male mice colonized with (i) the “core” community, (ii) the “core plus C. sporogenes” community, and (iii) the “core plus all” community. The pie charts depict the combined abundance of TMA-producing species and non-TMA-producing species in the community. The color bar chart (right) shows the partial contribution of each TMA producer to the total TMA-producing fraction in “core plus all” community. A. hydrogenalis was not detectable in the cecal samples of mice colonized with the “core plus all” community (10,000 to 60,000 reads/sample). (B to E) TMA abundance (in arbitrary units) in cecum (B), serum levels of TMAO (C), fecal levels of choline (D), and (E) serum levels of choline in mice colonized with various communities. Values are averages plus standard errors of the means (SEMs) (error bars) (4 or 5 animals in each experimental group). Values that were significantly different by an unpaired two-tailed Student’s t test are indicated by a bar and asterisk as follows: *, P value of <0.05; **, P value of <0.01; ****, P value of <0.0001. Similar results were observed in adult female mice (i.e., TMA and TMAO levels were detected only when animals were colonized with TMA-producing bacteria).

Gender modulates TMAO accumulation in serum. (A to D) Serum TMAO levels (A), cecal TMA abundance (B), total hepatic FMO enzymatic activity levels (C), and microbial community composition in cecal contents from male and female adult NMRI mice that were colonized with the “core plus all” community () (D) and maintained on a choline-supplemented diet (4 or 5 mice in each experimental group). Samples with less than 10,000 reads were not used for analysis. COPRO-Seq results shown in the pie charts are the average abundance of TMA-producing species and non-TMA-producing species in the community. The color bar charts show the partial contribution of each TMA producer to the total TMA-producing fraction. Statistical significance was calculated by an unpaired two-tailed Student’s t test and indicated by a bar and asterisks as follows: *, P value of <0.05; ****, P value of <0.0001.

Dietary choline is required for TMAO accumulation. (A) Levels of TMAO in serum from adult male C57BL/6J mice colonized with the “core plus all” community () and fed a 1% (wt/wt) choline-supplemented diet or a choline-deficient diet for 2 weeks after colonization. Data shown are averages plus SEMs (3 mice per group). Similar results were observed when the experiment was conducted in NMRI mice (4 or 5 mice per group; see in the supplemental material). (B) COPRO-Seq analysis of cecal contents from the mice described above for panel A. Samples included in the analysis have >10,000 reads. The pie charts depict combined abundance of TMA-producing species and non-TMA-producing species in the community. The color bar charts show the partial contribution of each TMA producer to the total TMA-producing fraction. Values that were statistically significant (P value of <0.01) by an unpaired two-tailed Student’s t test are indicated (**).